Vibrio vulnificus (V. vulnificus) is a bacterium in the same family as those that cause cholera. It normally lives in warm seawater and is part of a group of vibrios that are called "halophilic" because they require salt.
V. vulnificus can cause disease in people who eat contaminated seafood or have an open wound that is exposed to seawater. (Blake et al., Disease caused by a marine Vibrio: clinical characteristics and epidemiology, N. Eng. J. Med. 300:1-5 (1979) Among healthy people, ingestion of V. vulnificus can cause vomiting, diarrhea, abdominal pain and wound infections. It has been associated with severe wound infections or septicemia, particularly in immunocompromised individuals and in persons with chronic liver disease, where it can cause a severe life-threatening illness characterized by fever and chills, decreased blood pressure (septic shock), and blistering skin lesions. Death can occur in as little as three days after eating raw or improperly cooked fish or shellfish that are infected with V. vulnificus. In the United States this pathogen contributes to the majority of deaths associated with the consumption of seafood. (Rippey, Infectious diseases associated with molluscan shellfish consumption, Clin. Microbiol. Rev. 7:419-25 (1994)) V. vulnificus bloodstream infections are fatal about 50% of the time. Disease is highly correlated with the ingestion of raw oysters, which are frequently colonized by numbers of V. vulnificus exceeding 10.sup.4 bacteria/g wt. (Wright et al., Distribution of Vibrio vulnificus in the Chesapeake Bay, Environ. Microbiol. 62: 717-24 (1996)). One-third of persons with septicemia present with shock, and mortalities generally exceed 50%, although the mortality rate among patients who are hypotensive within 24 hours of hospital admission exceeds 90%.
To date, effective and commercially suitable assays specific for virulent strains of V. vulnificus, are not known in the art, and there has been a need to provide a means to detect and/or diagnose humans or animals infected with virulent strains of V. vulnificus. This bacterium is ubiquitous to the estuarine environment world wide, and in summer months virtually all oysters from the Atlantic and Gulf of Mexico are contaminated with this organism. Thus, there is no way to certify oysters as being pathogen-free, and the oyster industry in Southern states has suffered tremendous loses as many states will no longer accept Gulf Coast oysters. Warnings about the health risks related to V. vulnificus disease are now required for all oyster products in the U.S.
Traditional tests for the presence of V. vulnificus in food and water, as well as in patient samples (stool, blood, etc.), require the bacterium to be grown or cultured in a growth medium and examined morphologically and biochemically, a process that can take as long as one week. Such a procedure is ineffective when a patient requires a rapid diagnosis, as infection can be fatal in as little as three days. Similarly, such a process can not practically be applied to seafood safety testing which requires large shipments of seafood to be examined in a fast and inexpensive manner.
A faster way to identify the presence of these microbes is to use an antibody that recognizes and specifically binds to a specific part of the bacterium. See, for example, Tamplin, M. L. et al.: Enzyme immunoassay for identification of Vibrio vulnificus in seawater, sediment, and oysters, Applied and Environmental Microbiology, 57:1235-1240 (April 1991), which describes an enzyme immunoassay for the identification of V. vulnificus in seawater, sediment and oysters. Also, DNA probes for detecting this pathogen are described in U.S. Pat. No. 5,258,284 to Morris et al., and U.S. Pat. No. 5,607,835 to Reeves et al.
A key disadvantage of these known probes and antibodies is that they detect both virulent and non-virulent strains of V. vulnificus. The virulence of this microbe is linked to expression of capsular polysaccharide (CPS) on the surface of the bacterium. These capsules function to envelop the bacteria in a protective coating that is required for the evasion of host innate immune defenses such as phagocytosis and complement-mediated cell lysis. Hence, those bacteria that produce life-threatening systemic infections usually express CPS which can be a target for specifically detecting or attacking these harmful bacterium. Unfortunately, many pathogens can exhibit multiple types of CPS, which may confound identification and reduce or eliminate vaccine efficacy. On the other hand, CPS export systems are more highly conserved among species yet retain a high degree of species-specificity. Thus, these transport systems are potential targets for probes that are both species-specific and virulence specific.
In addition to the need for more specific diagnostics, there is also a need for improved vaccines and therapeutics for V. vulnificus exposure. Infection is usually treated with antibiotics such as doxycycline or cephalosporin. However, the rapid progression and high mortalities associated with this disease indicate that the current antibiotic therapies are ineffectual. A vaccine for this disease is currently not available, and reliable methods to eliminate V. vulnificus from oysters do not exist.
For the foregoing reasons, it would be desirable to have DNA probes and antibodies that are specific for only virulent forms of V. vulnificus and to develop improved vaccines and therapeutics for V. vulnificus exposure.